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The Role of Bmp4 and Bmp8b proteins in genesis of mouse primordial germ cells
Vegrichtová, Markéta ; Tlapáková, Tereza (advisor) ; Petelák, Aleš (referee)
Primordial germ cells are precursors of sexual breeding organisms. For induction of their formation are necessary growth factors from extraembryonic ectoderm. There are key proteins BMP4 a BMP8b among these factors. These proteins form both homodimers and heterodimers and through receptor complexes and SMAD proteins are indicating the competent cells of proximal epiblast, which give rise to the primordial germ cells and other structures of embryonic mezoderm. Although BMP4 and BMP8b proteins signal similarly, they are mutually indispensable and though for induction of primordial germ cells are both necessary. Up to now, it was proposed several models of their interaction, but not yet completely accurate.
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Effect of the sperm ubiquitination in the early embryonic development in pig
Petelák, Aleš ; Krylov, Vladimír (advisor) ; Petr, Jaroslav (referee)
The intracellular sperm injection (ICSI) technique is a very effective tool for the fertilization research. In the newly established laboratory at the Faculty of Science of the Charles University it was necessary to introduce this method and define the early developmental potential of fertilized oocytes. After fertilization oocytes were incubated to the blastocyst stage with a success comparable with other laboratories (17%). The ubiquitin-proteasome system which plays a major role in a protein degradation within cells is involved in a regulatory mechanism of sperm maturation. It is also responsible for a penetration of a vitelline membrane. In these processes ubiquitin residues are localized extracellulary. High level of sperm ubiquitination correlates with their low quality. Hypotetically it can be expected that the ubiqutination of impaired sperm cells can be used as a negative marker for their recognition and degradation by 26S proteasome complex localized. Experiments in this diploma thesis were designed based on the hypothesis that the executive part of the selective mechanism is the 26S proteasome. Therefore the effect of MG132 peptide inhibition of the 20S proteasome on the pronuclei formation and subsequent early embryonic development after ICSI was studied. Inhibition of 20S proteasome...
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Effect of sperm ubiquitination in early embryonic development of porcine embryos
Petelák, Aleš
The PhD thesis is focused on the effect of porcine sperm cell extracellular ubiquitination on early embryonic development up to the blastocyst stage after ICSI. In addition, it also presents a potential improvement of the technique of in vitro fertilization using oocyte incubation with ion channels regulators. To address these aims, we established an entirely novel methodology for sperm cell sorting using flow cytometry and subsequent cryopreservation. We determined the conditions for successful sperm cell sorting based on extracellular ubiquitination rate providing highly specific selection as well as sufficient numbers of viable sperms for fertilization using the ICSI method. Concerning the following cryopreservation, established methods were optimized to enable freezing of a minimal sperm cell suspension volume with low cell numbers. The performed experiments showed a direct relationship between the rate of extracellular ubiquitination and the capability of sperms to give rise to a properly developing embryo. Highly ubiquitinated sperm cells were less successful regarding the embryonic development to the blastocyst stage if compared with the lowly ubiquitinated group (6,2 % vs. 16,7 %, P<0,001). Interestingly, the rate of extracellular ubiquitination showed no effect on the pronuclear formation...
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Effect of sperm ubiquitination in early embryonic development of porcine embryos
Petelák, Aleš ; Krylov, Vladimír (advisor) ; Šušor, Andrej (referee) ; Procházka, Radek (referee)
The PhD thesis is focused on the effect of porcine sperm cell extracellular ubiquitination on early embryonic development up to the blastocyst stage after ICSI. In addition, it also presents a potential improvement of the technique of in vitro fertilization using oocyte incubation with ion channels regulators. To address these aims, we established an entirely novel methodology for sperm cell sorting using flow cytometry and subsequent cryopreservation. We determined the conditions for successful sperm cell sorting based on extracellular ubiquitination rate providing highly specific selection as well as sufficient numbers of viable sperms for fertilization using the ICSI method. Concerning the following cryopreservation, established methods were optimized to enable freezing of a minimal sperm cell suspension volume with low cell numbers. The performed experiments showed a direct relationship between the rate of extracellular ubiquitination and the capability of sperms to give rise to a properly developing embryo. Highly ubiquitinated sperm cells were less successful regarding the embryonic development to the blastocyst stage if compared with the lowly ubiquitinated group (6,2 % vs. 16,7 %, P<0,001). Interestingly, the rate of extracellular ubiquitination showed no effect on the pronuclear formation...
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Effect of sperm ubiquitination in early embryonic development of porcine embryos
Petelák, Aleš ; Krylov, Vladimír (advisor) ; Šušor, Andrej (referee) ; Procházka, Radek (referee)
The PhD thesis is focused on the effect of porcine sperm cell extracellular ubiquitination on early embryonic development up to the blastocyst stage after ICSI. In addition, it also presents a potential improvement of the technique of in vitro fertilization using oocyte incubation with ion channels regulators. To address these aims, we established an entirely novel methodology for sperm cell sorting using flow cytometry and subsequent cryopreservation. We determined the conditions for successful sperm cell sorting based on extracellular ubiquitination rate providing highly specific selection as well as sufficient numbers of viable sperms for fertilization using the ICSI method. Concerning the following cryopreservation, established methods were optimized to enable freezing of a minimal sperm cell suspension volume with low cell numbers. The performed experiments showed a direct relationship between the rate of extracellular ubiquitination and the capability of sperms to give rise to a properly developing embryo. Highly ubiquitinated sperm cells were less successful regarding the embryonic development to the blastocyst stage if compared with the lowly ubiquitinated group (6,2 % vs. 16,7 %, P<0,001). Interestingly, the rate of extracellular ubiquitination showed no effect on the pronuclear formation...
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The impact of intracytoplasmic sperm injection on succesful development and fertility in selected mammal species
Kaufman, Jonáš ; Petelák, Aleš (advisor) ; Petr, Jaroslav (referee)
5 Abstract This thesis aims to summarize the current knowledge about the development and reproductive capacities of individuals conceived by artificial conception, especially intracytoplasmic sperm injection (ICSI), and to make a comparison with naturally conceived ones. The focus is primarily on human assisted reproduction, even though scientific work with human gametes and embryos faces ethical difficulties. However, the main objective is to determine whether ICSI has a negative effect on offspring, partially using results from animal models. Furthermore, this thesis compares birth outcomes, incidence of birth defects, cognitive and reproductive abilities and both genetic and epigenetic integrity of individuals conceived naturally with those conceived via ICSI. As of yet, according to available information, ICSI appears to be a safe artificial method of reproduction. Poor results may often either be explained by the reduced fertility of parents, or their significance is negligible. Keywords: artificial fertilization, ART, ICSI, reproduction, offspring, mouse, livestock, human
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Role of tetraspanin network during sperm-egg interaction and fusion
Černá, Pavlína ; Hortová, Kateřina (advisor) ; Petelák, Aleš (referee)
Fusion of sperm and egg is a crucial moment of the fertilization process which is fundamentally urged by tetraspanin superfamily proteins whose interactions allow mutual interaction of gamets and their subsequent fusion. Tetraspanins are 25-50 kD transmembrane proteins whose various kinds are found in most mammalian cells. Four transmembrane domains containing key amino acid residues indicating their function are their specificity. The essential feature of tetraspanins is their ability to form membranes lateral bonds which are reached thanks to large complexes with other proteins that form the so-called tetraspanin network. The interaction with other proteins and tetraspanins is important for a cell adhesion to the extracellular matrix, adhesion to the other cells, and it plays a role in signal transduction. This bachelor thesis describes and summarizes the role of tetraspanin networks during fusion of egg and sperm. A general introduction of tetraspanins, their structure and functions in cellular processes and interactions leading to the formation of tetraspanin networks which are the key point in fertilization, are given. A description of the fertilization process itself, the protein interactions and changes which precede it in included. The latest findings of the topic from the viewpoint of...
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Reproductive and epigenetic parameters in sperm connected to the disruptive development and early embryo loss.
Flintová, Jennifer ; Hortová, Kateřina (advisor) ; Petelák, Aleš (referee)
DNA methylation, histone modifications and regulation by non-coding RNAs are considered to play vital role in embryonal development and gametogenesis. Epigenetic mechanisms are not only inwardly programmed, but are massively affected by numerous exogenous aspects. Environmetal pollution and unhealthy parental lifestyle are two major factors related to impaired fertility status, abberant embryonal development of progeny. Some altered epigenetic marks can be transmitted to offspring generations, however in some cases these aberrations may be reversed by adequate lifestyle interventions. Since epigenetic regulations and genes responsible for spermatogenesis are conserved among vertebrates, studies based on animal models are highly relevant for understanding mechanisms causing impaired fertility and overall health in humans. Key words: Epigenetics, sperm, endocrine disruptors, hypoxia, lifestyle, transgenerational effect.
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Chemical communication of gametes
Otčenášková, Tereza ; Komrsková, Kateřina (advisor) ; Petelák, Aleš (referee)
Many diverse chemoattractants play a part in sperm guidance towards the oocyte without any difference whether organisms with external or internal fertilization. Upon release, spermatozoa are exposed to various cues providing essential information about their surrounding environment. These molecules are structurally very distinct and they induce via specific receptors, expressed over the sperm cell surface, different signalling pathways influencing the sperm motility and regulating the correct sperm function. The overall understanding of these mechanisms brings interesting and often unexpected points of view on evolutionary relationships between invertebrate and vertebrate species. Some principles believed to be shown exclusively only by mammals are nowadays revealed also by marine invertebrates and vice versa. The current knowledge of human gametes interactions is used in fields of medicine that are connected to the reproduction. Thus, with a help of current knowledge, researches aim to shed more light into causes of infertility, as well as to develop novel contraceptives or enhance the rate of successful in vitro fertilizations.
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